1. Field of the Invention
The present invention relates to rapid, accurate and highly sensitive methods for detecting water born pathogens. Specifically, pathogens such as bacteria and/or their heat shock polynucleotides are immobilized on a surface within an assay structure. Electrolabeling molecules are then non-covalently attached to the pathogens. The electrolabeling molecules are then activated so as to generate a current that may be measured. The current generated is quantitativly and directly related to the concentration of the pathogens and accurately correlates thereto. While the present invention is particularly suitable for detection of Cryptosporidium parvum, it is also suitable for detecting a wide variety of pathogens, proteins, polynucleotides and a variety of other biological molecules.
2. Prior Art
Combining assays that utilize specific chemical interactions like noncovalent binding, such as hybridization and immunoassays, with electrochemical detection provide for a wide range of uses because highly specific and precise current measurements can be performed with simple instrumentation, using opaque device materials, and in colored and turbid samples minimizing prior pre-treatment procedures. The use of chemical compounds specific to the analyte, such as polynucleotide probes and antibodies, further reduces the need for pre-treatment procedures and facilitates accurate testing of very “dirty” samples.
General immunoassay procedures involve immobilization of the primary antibody (Ab, rat-anti mouse IgG), followed by exposure to a sequence of solutions containing the antigen (Ag, mouse IgG), the secondary antibody conjugated to an enzyme label (AP-Ab, rat anti mouse IgG and alkaline phosphatase), and p-aminophenyl phosphate (PAPp). The AP converts PAPp to p-aminophenol (PAPR, the “R” is intended to distinguish the reduced form from the oxidized form, PAP0, the quinoneimine) which is electrochemically reversible at potentials that do not interfere with reduction of oxygen and water at pH 9.0, where AP exhibits optimum activity. In addition, PAPR does not cause electrode fouling, unlike phenol whose precursor, phenylphosphate, is often used as the enzyme substrate. Although PAPR undergoes air and light oxidation, these are easily prevented on small scales and short time frames. Picomole detection limits for PAPR and femtogram detection limits for IgG achieved in microelectrochemical immunoassays using PAPp volumes ranging from 20 μl to 360 μL have been reported previously. In capillary immunoassays with electrochemical detection, the lowest detection limit reported thus far is 3000 molecules of mouse IgG using a volume of 70 μL and a 20 min assay time. Those skilled in the art will recognize the above described assay as a sandwich-type immunoassay and will appreciate that this is only one of many immunoassays. Alternatives include competitive binding immunoassays and immunoassays utilizing a more general physisabsorbing material other than a primary antibody.
Immunoassays are only one category of a very wide variety of surface immobilization chemical detection assays. Northern and southern blot assays are well known techniques for detecting specific polynucleotide sequences. They involve surface immobilization of polynucleotides. Surfaces having one or more lipid layers may be used to immobilize and detect compounds having hydrophobic regions. Molecular interactions may also be taken advantage of to develop surface immobilization chemical detection assays. When two molecules are known to bind to one another, one may be covalently attached to a substrate. The substrate is then exposed to a sample such that the other interacting molecule is given an opportunity to bind to the substrate bound molecule. The substrate is then rinsed leaving only bound analyte on the substrate. A number of detecting methods may then be applied to the surface. Detecting methods include using secondary antibodies as described above, detecting the bi-products of an enzymatic reaction characteristic of the analyte, spectroscopy, flourescence, or other methods known to those skilled in the art.
These assays generally require a laboratory setting. A person wishing to analyze a sample with one of the above described assays most usually sends the sample to a laboratory. Even while in a laboratory, many chemical detection assays take a relatively long period of time.
Cryptosporidium is a genus of protozoan parasite commonly found in the gastrointestinal tract of vertebrates. There are eight named species of Cryptosporidium. C. parvum is infectious for 79 species of mammals, including humans, causing acute gastroenteritis. Unlike most parasites, C. parvum lacks host specificity among mammals and is able to cross-infect multiple host species.
All infections with Cryptosporidium are initiated by ingestion or inhalation of the oocyst. Because the parasite is transmitted in the form of an oocyst, oocysts have evolved to survive in harsh environmental conditions and are unusually resistant to natural stresses and chemical disinfectants. In addition, the presence of an exogenous oocyst encapsulating the protozoan parasite makes the parasite much more resistant to conventional water treatment processes. Measures to prevent or limit the spread of infection concentrate on eliminating or reducing infectious oocysts in the environment. For humans, disinfection procedures are sought to minimize person-to-person transmission and to deal effectively with contamination of water supplies.
Immunological techniques have been used to detect C. parvum in environmental specimens. The availability of monoclonal antibodies for specific antigens of Cryptosporidium facilitated development of these methods.
Immunofluorescence assays (IFA) are the most common assays used to detect Cryptosporidium oocytes in specimens and to detect the presence of a specific antibody. These methods employ fluorescent dyes which are combined with antibodies to make them fluoresce when exposed to ultraviolet light. In a typical IFA assay, water is filtered through a polypropylene cartridge filter or a flat, membrane filter. Both filters yield filtrates that are then subjected to purification before analysis by microscopy. The filtrate is removed from the filter and then centrifuged. Extraneous debris is removed by flotation over a sucrose solution. The supernatant is labeled with a fluorescein conjugated antibody against Cryptosporidium and examined by epifluorescence microscopy.
Some commercial immunofluorescent assays and reagents used to detect Cryptosporidial oocytes include: (1) HydroFluor Combo, an immunofluorescent assay system based on an oocyst-specific monoclonal antibody (IgM, OW3) (2) Detect IF Cryptosporidium, an immunofluorescent assay system based on an oocyte-specific monoclonal antibody (IgM, C1), and (3) Crypto IF Kit, an immunofluorescent assay system based on an oocyst-specific monoclonal antibody.
The disadvantages of immunofluorescence assays include their low recovery efficiency, long processing times, the need for highly trained analysts, high cost, the inability to discriminate viable or virulent strains and cross-reactivity of the probes with similar size and shaped algae. In addition, IFA detection often involves the time consuming and skill intensive step of looking at water sludge under a microscope for oocysts that have been labeled with a fluorescent antibody. It is also often difficult to distinguish oocysts from debris bound non-specifically by the antibodies. The procedure is expensive and often takes days to complete.
Enzyme-linked immunosorbent assays (ELISA) using oocyte-reactive monoclonal antibodies are also used to detect Cryptosporidium in contaminated water samples. Two basic ELISA tests have been used in the past for detecting Cryptosporidium antigen in samples: (1) the double antibody sandwich technique for the detection of antigens, and (2) the enzyme-linked indirect immunosorbent assay for the detection of antibodies.
In the double antibody sandwich method, antiserum is adsorbed to a well. Test antigen is added and, if complementary, binds to the antibody. An enzyme-linked antibody specific for the test antigen then binds to the antigen, forming a sandwich. The enzyme's substrate is then added, and the reaction produces a visible color change. In the indirect immunosorbent assay, an antigen is adsorbed to a well. Test antiserum is then added, with complementary antibody binding to the antigen. Enzyme-linked anti-human gamma globulin is then added. It binds to the bound antibody. The enzyme's substrate is then added, producing a visible color change.
Detection assays based upon polymerase chain reactions (PCR) have also been used to detect oocysts in clinical or environmental samples. Several DNA and RNA regions of C. parvum have been sequenced and have been reported to be assay targets for parasite detection.
Flow cytometry is another method used to detect parasitic contamination of water samples. Flow cytometry techniques can quantify whole oocysts but involves much preparation, and time and require extremely expensive equipment.
Numerous problems are associated with prior art methods of detecting Cryptosporidium in water and environmental samples. In addition to those mentioned and the general lack of precise, recitable assays, prior art techniques generally require that samples be transferred to a laboratory or to another remote location for the conduct of the assay. Prior art techniques lack the requisite reliability, speed and sensitivity to accurately detect Cryptosporidium in contaminated water samples.
The detection of infectious C. parvum oocysts in water and other environmental samples is essential to detecting and treating contaminated water supplies. It is crucial, therefore, that specific, rapid and highly sensitive assays be developed to detect the presence of the parasite accurately and reliably. The known methods of enzyme immunoassays and immunofluorescence do not fulfill these requirements. The source, viability and pathogenicity of oocysts found in water or other environmental samples cannot be reliably determined using prior art methods. There is a need for routine epidemiological surveillance and environmental monitoring that can be conducted on site to provide early detection of the parasite.
It is therefore desirable to provide a method for rapid chemical detection.
It is also desirable to provide a highly sensitive method for detecting low amounts of analyte in a very small amount of sample.
It is also desirable to provide a method for detecting an analyte in a small sample having very high accuracy.